Alcohol is an important teratogen. Much research has focused on the sensitivity of the third- trimester period of brain development, where ethanol has been shown to be neuro-toxic. However, we know very little about the vulnerability of the second trimester period. The second trimester is important, because during this trimester, fetal neural stem (NSCs) and progenitor (NPCs) cells give rise to most neurons of the adult brain. Previously, we showed that ethanol did not kill fetal cortical NSCs/NPCs. Rather, ethanol induced cell proliferation, but depleted NSCs and NPCs, suggesting that ethanol promoted aberrant NSC maturation. Importantly, ethanol suppressed four microRNAs; small non-coding RNA molecules that control large gene networks to determine cell fate. Our central hypothesis is that ethanol depletes NSCs by driving stem to blast maturation of NSCs. A secondary hypothesis is that microRNAs mediate teratogenic effects of ethanol. We will address three specific aims. (Aim#l) To determine the extent to which ethanol promotes premature cortical neuroepithelial maturation. Our hypotheses are that (a) in utero binge ethanol exposure, during the second trimester period will significantly deplete resident NSCsINPCs and (b) isolated NSCs/NPCs will exhibit aberrant maturation patterns following ethanol exposure. We will test the ethanol- sensitivity of NSC/NPC populations using flow cytometric approaches. (Aim#2) To identify ethanol- sensitive microRNAs and their biological mechanisms. Our hypothesis is that ethanol persistently suppresses microRNAs that promote NSC/NPC renewal. We will use microarray technologies to identify candidate microRNAs, and manipulate levels of these microRNAs, to identify their role in NSC/NPC maturation. (Aim#3) To determine the extent to which ethanol-sensitive microRNAs prevent or reverse the effects of ethanol on fetal NSCs/NPCs. Our hypothesis is that microRNA supplementation will prevent and reverse ethanol's effects on NSC/NPC renewal and maturation. Cell biological approaches and mRNA expression analyses and will be used to assess the capacity of ethanol-regulated microRNAs to reverse or prevent the effects of prior ethanol exposure. At the end of the project period, we expect to have identified sensitive NSC/NPC populations, critical second-trimester periods of vulnerability to ethanol, and specific microRNAs and microRNA- mediated mechanisms that are persistently altered by ethanol. These outcomes will be significant because they are expected to provide the foundations for developing therapeutic strategies to manage the persistent neural effects of a leading teratogen. PUBLIC HELTH RELEVANCE Alcohol is a potent teratogen. Alcohol consumption during pregnancy can lead to a stereotypic spectrum of disorders in children, called the `Fetal Alcohol Spectrum Disorders' or FASD. At the neuroanatomical level, birth defects can include microencephaly and brain malformations. Much research has focused on the sensitivity of the third-trimester period of brain development, where ethanol has been shown to be neurotoxic. However, we know very little about the vulnerability of the second trimester period. The second trimester is important, because during this trimester, neuroepithelial cells of the ventricular walls give birth to millions of new neurons, creating a cellular framework for the rest of neural development. Previously, we showed that fetal cerebral cortical neuroepithelial cells did not die when exposed to ethanol. Rather, ethanol induced cell proliferation, while depleting cells expressing neural stem (NSC) and progenitor cell (NPC) markers. This proposal will test two hypotheses, that ethanol depletes stem and progenitor cells by driving stem to blast maturation of NSCs, and that the effects of ethanol are mediated by, and consequently, can be reversed by microRNAs. At the end of the project period, we expect to have identified sensitive NSC/NPC populations, critical second-trimester periods of vulnerability to ethanol, and specific microRNAs and microRNA-mediated mechanisms that are persistently altered by ethanol. These outcomes will be significant because they are expected to provide the foundations for developing therapeutic strategies to manage the persistent neural effects of a leading teratogen.